Nucleic acid probe technology has developed rapidly in recent years as researchers have discovered its value for detection of various diseases, organisms or genetic features which are present in small quantities in a human or animal test sample. The use of probes is based upon the concept of complementarity such that complementary strands of nucleotides can and will, under proper conditions, hybridize to form a double stranded product. DNA inherently has two strands bound together by hydrogen bonds between complementary nucleotides (which are also known as nucleotide pairs). RNA, while being single stranded, also can hybridize with a complementary strand of nucleotides.
Hybridization of complementary strands is a central feature of what are known as hybridization assays (also known as genetic probe assays). Thus, if at least a sequence of a nucleic acid of interest (identified as a target nucleic acid) is known, it can be hybridized and detected using appropriate oligonucleotides (identified as probes) which are designed to be complementary to that known sequence. The probe usually is labeled in some fashion so that its detection also signals the presence of the hybridized product of probe and target nucleic acid. If the target nucleic acid is then unique to a particular organism, viral infection or genetic feature, the presence of the organism, virus or genetic feature can then readily be determined.
In some instances, the target nucleic acid (single strand form) is insolubilized on a solid surface (such as a nitrocellulose membrane) to prevent undesirable hybridization with oligonucleotides other than probes. Detection of the immobilized target nucleic acid can be accomplished using a detection probe specific for the target nucleic acid. Some have pointed out the disadvantages of such systems, for example EPA-0 318 245 (published May 31, 1989).
In what are known as "immunometric" or "sandwich" assays, the target nucleic acid may be captured using another oligonucleotide which is immobilized in some manner, for example on a membrane as described in U.S. Pat. No. 4,727,019 (issued Feb. 23, 1988). In other instances, the oligonucleotide is immobilized on polymeric particles which are embedded within a porous matrix (for examples as described in EP-A-0 200 381, published Nov. 5, 1986). The immobilized oligonucleotide is sometimes identified as a capture probe meaning that it "captures" the target nucleic acid so that it can be separated from undesirable materials which might obscure its detection. Once separation is accomplished, detection of the captured target nucleic acid can be achieved using a suitable procedure.
It is well known to attach oligonucleotides to particulate materials in hybridization assays and for purification of nucleic acids. EPA-0 200 133 (published Nov. 5, 1986), for example, describes the attachment of oligonucleotides to water-insoluble particles less than 50 micrometers in diameter for use in hybridization assays. Various linking groups for attaching nucleic acids to glass, polystyrene and latex particles are described in WO 88/01302 (published Feb. 25, 1988). This reference also mentions the use of dextran sulfate or a "non-homologous" nucleic acid to treat the capture probe prior to its use to increase capture efficiency. A "non-homologous" nucleic acid is defined as one that is "inert" which is believed to mean that it is not complementary to the probe oligonucleotide.
EP-A-0 318 245 (noted above) describes the need to improve the kinetics of hybridization between a water-soluble probe (such as a detection probe) and a long target nucleic acid in solution hybridization assays. This need is allegedly met using "helper" oligonucleotides which, it is believed, reorder the secondary and tertiary structure of the target nucleic acid in solution. These oligonucleotides hybridize with the target nucleic acid in regions other than that where the detection probe hybridizes.
Where a capture probe is used in a hybridization assay, the oligonucleotide attached to a water-insoluble support (such as a small polymeric particle) is usually very small in relation to the size of the support. The length of the oligonucleotide is so short that it extends only a very short distance from the surface of the support. Thus, steric hindrance is believed responsible for slow hybridization rates between the capture probe and the target nucleic acid.
One technique for solving this problem is described in U.S. Ser. No. 197,000 (filed May 20, 1988 by Saiki et al), abandoned. Improved hybridization is apparently achieved by inserting a polyribonucleotide or polydeoxyribonuecleotide spacer group between the support and the short capture oligonucleotide to extend the oligonucleotide away from the support surface.
Thus, there is a continuing need to improve the hybridization efficiency between target nucleic acids and insolubilized capture probes having very short oligonucleotides.